RNA used to be considered a simple and straightforward molecule in cells. The three major classes of RNA, i.e., transfer RNA, ribosomal RNA, and messenger RNA (mRNA), have generally not been thought to be subjected to regulation by signaling pathways, or to have major roles in disease processes. However, a rapidly emerging concept over the past few years is that transcription and other cell signaling pathways are regulated by a diverse array of noncoding RNAs, such as microRNAs, termini-associated RNAs (Han et al., “Promoter-associated RNA Is Required for RNA-directed Transcriptional Gene Silencing in Human Cells,” Proc Natl Acad Sci USA 104:12422-12427 (2007)), and other noncoding RNAs. Additionally, mRNA is no longer viewed as a simple intermediate between DNA and protein, but instead is now known to be subjected to wide range of post-transcriptional processing events, including diverse types of splicing reactions, nonsense-mediated decay, RNA editing, exo- and endonucleolytic degradation, polyadenylation, and deadenylation. Another intriguing aspect of RNA biology is the finding that trinucleotide repeat-containing mRNAs exert specific gain-of-function toxicities associated with their accumulation at certain intracellular sites (Ranum et al., “Myotonic Dystrophy: RNA Pathogenesis Comes Into Focus,”Am. J. Hum. Genet. 74:793 (2004)). In addition to these different regulatory pathways, recent studies indicate that RNAs traffic through different parts of the cell during RNA maturation. For example, nascent RNA transcripts are likely trafficked to specific intracellular sites in the nucleus for processing events, such as splicing, nonsense-mediated decay, or for packaging into transport granules. After nuclear export, some RNAs have been localized to RNA-enriched intracellular structures including RNA granules, stress granules, and processing bodies (P-bodies) (Kiebler et al., “Neuronal RNA Granules: Movers and Makers,” Neuron 51:685-690 (2006)). The diversity of these RNA regulatory mechanisms makes it clear that RNA is regulated by a complex and intricate network of regulatory mechanisms and intracellular structures that have a critical role in gene expression.
RNA is increasingly being utilized for various biotechnology applications, including as sensors (Breaker, “Engineered Allosteric Ribozymes as Biosensor Components,” Curr Opin Biotech. 13:31 (2002); Cho et al., “Applications of Aptamers as Sensors,” Annu Rev Anal Chem. 2:241 (2009)), nanodevices (Sherman and Seeman, “Design of Minimally Strained Nucleic Acid Nanotubes,” Biophys. J. 90:4546 (2006); Win et al., “Frameworks for Programming Biological Function through RNA Parts and Devices,” Chem. Biol. 16:298 (2009)), catalysts (Joyce, “Directed Evolution of Nucleic Acid Enzymes,” Annu. Rev. Biochem. 73: 791 (2004); Lincoln and Joyce, “Self-sustained Replication of an RNA Enzyme,” Science 323:1229 (2009)), protein inhibitors (Lee et al., “Aptamer Therapeutics Advance,” Curr. Opin. Chem. Biol. 10:282 (2006)), and in the development of supramolecular structures (Chworos et al., “Building Programmable Jigsaw Puzzles with RNA,” Science 306:2068 (2004); Dirks et al., “Paradigms for Computational Nucleic Acid Design,” Nucleic Acids Res. 32:1392 (2004); Levy-Nissenbaum et al., “Nanotechnology and Aptamers: Applications in Drug Delivery,” Trends Biotechnol. 26:442 (2008)). The ability to confer GFP-like functionality to RNA will facilitate molecular studies of RNA and advance various RNA-based applications.
Although PCT Application Publ. No. WO/2010/096584 to Jaffrey and Paige describes a number of RNA aptamers that bind to conditionally fluorescent molecules derived from the chromophore of green fluorescent protein, and their use, for example, in cellular imaging and RNA trafficking, there continues to be a need for improved aptamers and aptamer-fluorophore complexes to enhance the generation of aptamer-based small molecule sensors as well as in vitro and in vivo monitoring of RNA molecules.
The present invention is directed to overcoming these and other deficiencies in the art.